GILZ Overexpression Inhibits Endothelial Cell Adhesive Function through Regulation of NF-κB and MAPK Activity

Qiang Cheng, Huapeng Fan, Devi Ngo, Elaine Beaulieu, Patrick Leung, Camden Y. Lo, Rosemary Burgess, Yvonne G. van der Zwan, Stefan J. White, Levon M. Khachigian, Michael J. Hickey and Eric F. Morand
J Immunol July 1, 2013, 191 (1) 424-433; DOI: https://doi.org/10.4049/jimmunol.1202662

Abstract

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory protein first identified in T lymphocytes. We recently observed that GILZ is highly expressed in synovial endothelial cells in rheumatoid arthritis. However, the function of GILZ in endothelial cells is unknown. To investigate the actions of GILZ in this cell type, we induced GILZ expression in HUVECs via transient transfection. GILZ overexpression significantly reduced the capacity of TNF-stimulated HUVECs to support leukocyte rolling, adhesion, and transmigration. These effects were associated with decreased expression of E-selectin, ICAM-1, CCL2, CXCL8, and IL-6. Experiments in a human microvascular endothelial cell line demonstrated that TNF-inducible NF-κB activity was significantly inhibited by overexpression of GILZ. Exogenous GILZ inhibited TNF-induced NF-κB p65 DNA binding, although this occurred in the absence of an effect on p65 nuclear translocation, indicating that the mechanism of action of exogenous GILZ in endothelial cells differs from that reported in other cell types. GILZ overexpression also inhibited TNF-induced activation of p38, ERK, and JNK MAPKs, as well as increased expression of the MAPK inhibitory phosphatase, MKP-1. In contrast, silencing endogenous GILZ in glucocorticoid-treated HUVECs did not alter their capacity to support leukocyte interactions. These data demonstrate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function via a novel pathway involving modulation of NF-κB p65 DNA binding and MAPK activity. Induction of GILZ expression in endothelial cells may represent a novel therapeutic modality with the potential to inhibit inflammatory leukocyte recruitment.

Introduction

Leukocyte recruitment from the bloodstream in response to inflammatory stimuli is an essential step in host defense against pathogens and in wound healing. However, this process is also fundamental to the pathogenesis of inflammatory disease (1). To leave the circulation and be recruited to sites of inflammation, leukocytes undergo a sequence of interactions on the endothelium lining the microvasculature at sites of inflammation, initially rolling and then arresting on the endothelial surface before crawling across the endothelium to encounter an optimal site to leave the vasculature (2, 3). Via its ability to express adhesion molecules and chemokines that support these interactions, the vascular endothelium plays an integral role in inflammatory responses (1). At the same time, numerous endogenous anti-inflammatory molecules exist that restrict inflammation via actions on endothelial cells (4–6).

Glucocorticoid-induced leucine zipper (GILZ) is an anti-inflammatory molecule first identified in T lymphocytes as a glucocorticoid-induced protein that suppresses T cell activation (7). Subsequent studies showed that GILZ has the capacity to modulate several important proinflammatory-signaling pathways. Most prominently, studies in T lymphocytes demonstrated that GILZ inhibits the nuclear translocation of the p65 subunit of the transcription factor NF-κB, thereby reducing NF-κB transcriptional activity (8–11). GILZ was also observed to inhibit activation of the AP-1 transcription factor and to interact with Raf-1/Ras, leading to suppression of downstream ERK MAPK activation (12–14). The identification of GILZ as an important anti-inflammatory protein is supported by recent in vivo studies. We observed that inhibition of GILZ expression via systemically administered small interfering RNA (siRNA) increased disease severity in murine collagen-induced arthritis, via a mechanism associated with increased proinflammatory cytokine production by phagocytic cells (15). Concordantly, a recent study showed that administration of a GILZ peptide inhibits disease development in the experimental autoimmune encephalomyelitis model of multiple sclerosis, in part via effects on T cell activation (11). Together, these findings demonstrate a suppressive role of GILZ in the regulation of immune cell activation. However, it is unclear whether GILZ can inhibit proinflammatory responses in other cell types, such as endothelial cells.

We recently observed the expression of GILZ in synovial endothelial cells in rheumatoid arthritis (15), raising the possibility that the endothelial cell is an important site of action for GILZ in vivo. Therefore, the aim of this study was to examine the ability of GILZ to regulate proinflammatory endothelial cell function. We found that exogenous GILZ expression in human endothelial cells reduces their ability to support interactions with leukocytes under flow conditions, an effect associated with reduced adhesion molecule, cytokine, and chemokine expression, as well as inhibition of NF-κB and MAPK activation. However, no effect of silencing endogenous GILZ on the regulation of TNF-induced endothelial adhesive function was observed in the presence or absence of dexamethasone (DEX). These results suggest that exogenous GILZ expression is a previously unrecognized mechanism for inhibition of inflammatory activation of human endothelial cells.

Materials and Methods

Proteins, Abs, and chemicals

Recombinant human TNF was purchased from R&D Systems (Minneapolis, MN). PE-labeled mouse anti-human E-selectin mAb, PE-labeled mouse anti-human ICAM-1 mAb, and FITC-labeled mouse anti-human VCAM-1 mAb were all purchased from BD Biosciences (San Diego, CA). Bay 11-7082 (NF-κB inhibitor) was obtained from Calbiochem (Darmstadt, Germany).

Human endothelial cells

Primary HUVECs were isolated from umbilical veins and cultured in 0.2% gelatin-coated culture flasks with either complete M199 medium (Invitrogen, Carlsbad, CA) with 20% FBS or Endothelial Growth Medium (Lonza, Basel, Switzerland), as described previously (16). Human microvascular endothelial cells (HMEC-1; American Type Culture Collection, Manassas, VA) were grown in MCDB 131 medium (Invitrogen) containing 10% FBS, l-glutamine (2 mM), epidermal growth factor (10 ng/ml), hydrocortisone (1 μg/ml), and penicillin-streptomycin (5 U/ml) (Invitrogen), as described previously (17). To activate HMECs, cells were serum starved overnight before being treated with TNF in serum-free medium. For NF-κB–inhibition experiments, cells were treated with Bay 11-7082 (2 μM) for 1 h before the addition of TNF for 4 h.

Transient transfection in HUVECs

The human GILZ-expressing construct (pcDNA-GILZ) was a generous gift from Dr. Carlo Riccardi (Department of Clinical and Experimental Medicine, University of Perugia, Perugia, Italy). Control pcDNA or pcDNA-GILZ was transfected into HUVECs using a HUVEC Nucleofector Kit (Lonza), according to the manufacturer’s instructions. HUVECs were trypsinized from culture flasks, and 0.5 × 106 cells were used for each electroporation with 3 μg plasmid DNA. After electroporation, 0.25 × 106 cells were seeded, at 1 × 106 cells/ml, onto 3.5-cm culture dishes (Corning, Lowell, MA) coated with fibronectin (Roche Diagnostics, Castle Hill, NSW, Australia). Cells were then cultured overnight before being used in experiments.

Western blot analysis

Cells were lysed in cell lysis buffer (Cell Signaling Technology, Beverly, MA) containing phosphatase and protease inhibitors (Roche Diagnostics, Indianapolis, IN). Immunoblotting was performed using Abs directed against human GILZ (G-5; Santa Cruz Biotechnology, Santa Cruz, CA), NF-κB p65 (Abcam, Cambridge, U.K.), TATA-binding protein (TBP), MKP-1, β-actin, phosphorylated and total p38, ERK1/2, and JNK (all from Cell Signaling Technology), as described elsewhere (18). To separate nuclear from cytoplasmic proteins, the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL) were used according to the manufacturer’s instructions. Membrane blot densitometry was performed using the Odyssey system (LI-COR Biosciences, Lincoln, NE), as previously described (18). Densitometry ratios were normalized to either total protein content (in phosphorylation assays) or β-actin, and results are expressed relative to unstimulated control cells.

Flow chamber assay

The whole-blood flow chamber assay for assessment of leukocyte–endothelial cell interactions was performed as reported previously (16). Briefly, in experiments to assess leukocyte rolling and adhesion, HUVEC monolayers were treated or not with TNF (0.03 ng/ml) for 4 h. Blood collected from healthy volunteers was then diluted 1:10 in HBSS and perfused over the HUVEC monolayer for 5 min at a flow rate of 150 s−1. Subsequently, blood was replaced with HBSS to clear the field, 8–12 random fields (0.23 mm2/field) were recorded for 10 s each, and the numbers of rolling and adherent (static for >10 s) leukocytes were determined on playback analysis. To assess leukocyte transmigration, HUVEC monolayers were treated with TNF at 1 ng/ml for 4 h before flow chamber analysis. After 5 min of blood perfusion (250 s−1), six random fields were recorded for 60 s using time-lapse microscopy at a recording rate of one frame/s. Transmigrated leukocytes were identified as phase-dark migratory cells under the endothelial cell monolayer (19). All parameters (rolling, adhesion, transmigration) were expressed as cells/mm2. Transmigration efficiency was defined as the percentage of transmigrated cells relative to the sum of adherent and transmigrated cells.

Quantitation of mRNA expression by real-time PCR

Total mRNA was extracted with the RNeasy mini kit (QIAGEN, Cologne, Germany). cDNA was generated using random primers (7.5 μg/μl) and the SuperScript III reverse transcriptase (Invitrogen). The gene-specific primer sequences used for real-time PCR are listed in Table I. Real-time PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems) with Rotor-Gene 3000 (Corbett Research, Mortlake, NSW, Australia). The level of target gene expression was normalized against 18S rRNA expression, and results are expressed as the number of mRNA copies/106 18S rRNA copies (16).

Quantitation of cytokine and chemokine production

The concentrations of IL-6 and CXCL8 in HUVEC culture supernatants were measured using beads appropriate for selected cytokines in the Cytometric Bead Array Human Inflammatory Cytokines Kit (BD Bioscience), according to the manufacturer’s instructions. All bead samples were analyzed by a FACSCanto II Cell Analyzer (BD Bioscience), and the results were analyzed using FCAP Array software (Version 1.0.1; Soft Flow). Cytokine concentrations were determined by reference to the cytokine standards. CCL2 was detected in the supernatant by ELISA (BD Bioscience), according to the manufacturer’s protocol.

Stable transfection of HMECs

For stable transfection of HMECs, the pNFκB-Luc (Stratagene, Santa Clara, CA) NF-κB reporter construct and pcDNA or pcDNA-GILZ were first linearized by digestion with NdeI and PvuI restriction enzymes (both from New England BioLabs, Ipswich, MA), respectively. Subsequently, 3 μg linearized pcDNA or pcDNA-GILZ and 15 μg linearized pNFκB-Luc were cotransfected into 1 × 106 HMECs via electroporation using the ECM 830 electroporation system (BTX, San Diego, CA) under the following conditions: 300 V, 200 μs, eight pulses, 1 Hz. Cells were subsequently cultured in complete medium overnight before being selected with G418 (1 mg/ml; Sigma-Aldrich, St. Louis, MO). After 10–15 d of selection, to minimize clonal variability, multiple clones were pooled to generate HMEC lines stably expressing the NF-κB reporter construct plus either pcDNA or pcDNA-GILZ (20). For luciferase reporter assays, cells were lysed with 100 μl lysis buffer, according to the manufacturer's instructions (Promega, Madison, WI). After this, 20 μl of the cell lysate was mixed with 35 μl luciferase reagent (Promega) and analyzed for luciferase activity using a luminometer (Wallac; Perkin Elmer, Turku, Finland).

Construction and infection of GILZ–recombinant adeno-associated virus 5

As an alternative mode for induction of GILZ expression, GILZ-recombinant adeno-associated virus 5 (GILZ-rAAV), a gift from Drs. Scott Loiler (Arthrogen, Amsterdam, The Netherlands) and Margriet Vervoordeldonk (Division of Clinical Immunology and Rheumatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands) was used (21). Stably transfected NF-κB reporter HMECs, generated as described above, were plated in a 96-well plate (2 × 104/well) and allowed to adhere overnight. Cells were then infected with human mock control or GILZ-rAAV at 400,000 multiplicity of infection in Pen/Strep-free HMEC medium containing 0.4 μM doxorubicin (Sigma-Aldrich) for 6 h before replacing media with fresh complete HMEC medium. Cells were left for 72 h before stimulating with TNF (0.03 ng/ml; 4 h). Cell lysates were then collected for luciferase assay.

Chromatin immunoprecipitation assay

HMECs were treated or not with TNF (0.3 ng/ml; 1 h), and the chromatin immunoprecipitation (ChIP) assay was performed according to the low cell number ChIP protocol from Diagenode (Liege, Belgium), with minor modifications. In brief, 1 × 106 cells were cross-linked for 8 min by addition of formaldehyde to a final concentration of 1%, followed by neutralization with 1.25 M glycine. The cells were then lysed, and chromatin was sheared to ∼500-bp fragments using the Covaris sonicator under the following conditions: duty cycle, 20%; peak incident power, 200 W, cycles/burst, 200; and time, 600 s.

Protein A–coated Dynabeads (Invitrogen) were incubated with 5 μg anti-p65 Abs (Abcam) or anti-IgG Abs (Vector Laboratories, Burlingame, CA) and then combined with chromatin from 5 × 105 cells overnight on a rotating wheel. The immunoprecipitated material was washed, and DNA was purified using the iPure DNA purification kit (Diagenode), according to manufacturer’s instructions. Enrichment was calculated by real-time PCR using primer pairs designed to amplify the p65 binding sites within the promoter regions of CCL2, CXCL8, and VCAM-1. Another primer pair that amplifies an encoding region of the human β-actin gene (ACTB) was also included as a nonbinding control. Sequences for all primer pairs used for ChIP analysis are shown in Table I. First, the ΔCt value of each sample was calculated by subtracting the nonbinding control value from the value of p65 binding site. Next, the ΔCt values of p65 binding sites were further normalized by subtracting the ΔCt values of the corresponding anti-IgG controls (ΔΔCt). Finally, the relative promoter occupancy by p65 was presented as a percentage of the value of TNF-treated pcDNA cells.

Immunofluorescence microscopy

GILZ and p65 were detected in HMECs using a two-layer staining protocol. Cells were washed once with PBS and fixed for 10 min with 4% formaldehyde/PBS at room temperature before permeabilization with 100% ice-cold methanol for 10 min at −20°C. Cells were blocked with blocking buffer containing 10% normal donkey serum, 10% normal goat serum, and 0.3% Triton X in 1% BSA/PBS for 1 h at room temperature. Cells were incubated with the following primary Abs: rabbit anti-human NF-κB p65 (1:300; Abcam) and mouse anti-human GILZ (G-5; 1:250; Santa Cruz Biotechnology) overnight at 4°C. This was followed by incubation (1 h at room temperature) with secondary Abs: FITC-conjugated donkey anti-rabbit IgG (1:500; Santa Cruz Biotechnology) and Alex Fluor 568 goat anti-mouse IgG (1:500; Invitrogen). Nonspecific rabbit and mouse IgG Abs (Vector Laboratories) were used as negative controls. All Abs were diluted in blocking buffer, and cells were washed twice with PBS between incubations. The cells were then stained with DAPI, via coverslipping with ProLong Gold Antifade Reagent (Invitrogen), before being examined on a Nikon Eclipse Ti-E inverted confocal microscope.

Immunoprecipitation

HMECs (3 × 106) were treated or not with TNF (0.3 ng/ml) for 1 h before being lysed in a nondenaturing cell lysis buffer (Cell Signaling Technology). These whole-cell lysate samples were then incubated at 4°C overnight with Protein A/G PLUS Agarose (Pierce, Rockford, IL) and Ab complex, which was prepared by incubating 40 μl Protein A/G Plus Agarose with 5 μg either anti-p65 Ab (Abcam) or control rabbit IgG Ab (Vector Laboratories). These Ag–Ab complexes were then washed, and protein samples were recovered in protein loading buffer (4% SDS, 20% glycerol, 0.12 M Tris [pH 6.8], and 10% 2-ME) at 95°C for 5 min. Western blot was performed using both anti-p65 and anti-GILZ Abs. Anti-p105/50 Ab (Cell Signaling Technology) was also included as positive binding control.

GILZ silencing via siRNA

For GILZ silencing, a human GILZ-specific siRNA (sense, 5′-AACAGCUUCACCUGACAACGAtt-3′; antisense, 3′-ttUUGUCGAAGUGGACUGUUGCU-5′) was used (22). Control cells were treated with the following nontargeting scrambled control siRNA: sense, 5′-CACUCGACGCACGCCGCACtt-3′; antisense, 5′-GUGCGGCGUGCGUCGAGUGtt-3′. Both siRNAs were purchased from Invitrogen. siRNA was transfected into HUVECs using the HUVEC Nucleofector Kit (Lonza), and cells were used for experiments 2 d after transfection, as previously described (16). To test the effects of DEX on TNF-induced HUVEC activation, cells were treated with DEX (10−6 M) for 1 h before being exposed to TNF in the presence of the same dose of DEX.

Statistics

Significance was assessed by the Student t test, and a p value < 0.05 was considered significant. Where appropriate, paired analysis was performed.

Results

Effect of exogenous GILZ expression in endothelial cells on leukocyte–endothelial cell interactions

To investigate GILZ function in human endothelial cells, GILZ expression was induced in HUVECs by transient transfection of the pcDNA-GILZ construct. GILZ protein was robustly expressed in pcDNA-GILZ–transfected cells, above the level of basal GILZ expression observed in control (pcDNA) transfectants (Fig. 1A). A whole-blood flow chamber system was used to test the effect of exogenous GILZ on the ability of HUVECs to support leukocyte–endothelial cell interactions. In the absence of TNF, minimal leukocyte rolling and adhesion were observed (Fig. 1B, 1C), indicating that transfection alone did not activate HUVECs. In contrast, TNF induced substantial rolling and adhesion in control-transfected HUVECs. However, in GILZ-transfected cells, leukocyte rolling and adhesion were significantly decreased, by an average of 37 and 26%, respectively (Fig. 1B, 1C). In separate experiments, we compared leukocyte transmigration in control and GILZ-transfected HUVECs (Fig. 1D, 1E) and found that both total transmigration and transmigration efficiency were reduced significantly in GILZ-transfected cells.

FIGURE 1.
FIGURE 1.

GILZ-overexpressing endothelial cells support fewer leukocyte–endothelial cell interactions. (A) GILZ expression (measured by Western blot) in HUVECs transiently transfected (24 h) with either pcDNA (control) or pcDNA-GILZ constructs. β-actin is shown as loading control. (B–E) The ability of HUVECs transfected with either pcDNA or pcDNA-GILZ to support interactions with human leukocytes under flow conditions was assessed using a flow chamber assay, in untreated cells, and following TNF stimulation. Data are shown for rolling (B) and adhesion (C), as assessed using TNF at 0.03 ng/ml (4 h). Data represent mean ± SEM of n = 7 experiments/group. In separate experiments (TNF, 1 ng/ml, 4 h), leukocyte transmigration (D) and transmigration efficiency (E) were assessed. Data represent mean ± SEM of n = 3 experiments/group. *p < 0.05.

View this table:
Table I. Primer sequences for real-time PCR and ChIP analyses

Effects of exogenous GILZ on HUVEC expression of adhesion molecules

Expression of endothelial adhesion molecules is critical in the induction of leukocyte–endothelial cell interactions (2). Therefore, we next examined whether the decreased ability of GILZ-transfected HUVECs to support leukocyte rolling and adhesion was associated with reduced expression of adhesion molecules. As expected, TNF treatment increased HUVEC expression of E-selectin, ICAM-1, and VCAM-1 at the levels of both mRNA and cell surface–expressed protein (Fig. 2). However, compared with pcDNA control-transfected cells, TNF-inducible E-selectin mRNA and cell surface protein expression was moderately, but significantly, reduced in GILZ-overexpressing HUVECs (Fig. 2A, 2B). Exogenous GILZ expression also resulted in significant decreases in basal and TNF-induced ICAM-1 mRNA and protein expression (Fig. 2C, 2D). In contrast, no effect of GILZ overexpression was observed on the induction of VCAM-1, indicating that exogenous GILZ did not globally inhibit adhesion molecule gene expression (Fig. 2E, 2F). These effects were observed in the absence of any changes in expression of mRNA for TNFR1 and TNFR2 (data not shown), indicating that the mechanism for this effect was downstream of TNFRs.

FIGURE 2.
FIGURE 2.

GILZ-overexpressing endothelial cells show reduced expression of E-selectin and ICAM-1 but not VCAM-1. Expression of E-selectin, VCAM-1, and ICAM-1 in pcDNA (control)-transfected or pcDNA-GILZ–transfected HUVECs. Data are shown for mRNA (A, C, E) and protein (B, D, F) in the absence of stimulation (NC) or following stimulation with TNF (0.03 ng/ml). mRNA levels were measured by real-time PCR after 1 and 4 h of TNF treatment, and protein expression was determined using flow cytometry at 4 h. Data represent mean ± SEM of n = 5 experiments/group. *p < 0.05, **p < 0.01.

Effect of GILZ on HUVEC expression of CCL2, CXCL8, and IL-6

Proinflammatory chemokines and cytokines produced by endothelial cells play important roles in the regulation of leukocyte recruitment. Therefore, we also examined the effects of exogenous GILZ on HUVEC expression of these molecules. GILZ-transfected HUVECs showed moderately, but significantly, decreased basal and TNF-induced CCL2 mRNA expression relative to control-transfected cells (Fig. 3A), with similar effects seen for basal and TNF-induced CCL2 protein release (Fig. 3B). TNF-induced CXCL8 mRNA and protein were also significantly reduced in GILZ-transfected HUVECs (Fig. 3C, 3D). GILZ-transfected HUVECs also displayed a significant reduction in TNF-induced release of IL-6 (Fig. 3F), although, in this case, no effect on IL-6 mRNA expression was observed (Fig. 3E).

FIGURE 3.
FIGURE 3.

GILZ-overexpressing endothelial cells have reduced expression of CCL2, CXCL8, and IL-6. Expression of CCL2, CXCL8, and IL-6 in pcDNA (control)-transfected or pcDNA-GILZ–transfected HUVECs in the absence of treatment or following treatment with TNF (0.03 ng/ml, n = 5). Data are shown for mRNA (A, C, E) and protein (B, D, F) in the absence of stimulation (NC) or following stimulation with TNF (0.03 ng/ml). mRNA levels were measured by real-time PCR at 1 and 4 h of TNF treatment, and protein concentration in the supernatants was determined using either Cytometric Bead Array kits or ELISA at 4 h. Data represent mean ± SEM of n = 5 experiments/group. *p < 0.05, **p < 0.01.

Effect of exogenous GILZ on endothelial cell NF-κB transcriptional activity

Studies in other cell types demonstrated the capacity of transfected GILZ to reduce activation of the NF-κB pathway (8, 11). However, it is unknown whether this mechanism also operates in endothelial cells. To address this, we generated stably transfected NF-κB luciferase reporter HMEC lines, which were cotransfected with either pcDNA-GILZ or control pcDNA. GILZ expression was markedly induced in stably GILZ-transfected HMECs, relative to pcDNA-transfected control cells, in which only minimal expression of GILZ could be detected (Fig. 4A). Given the suppressive effects of exogenous GILZ on CCL2 and IL-6 expression observed in HUVECs, we examined whether GILZ had the same impact on these NF-κB–dependent genes in HMECs. TNF significantly induced CCL2 expression at both the mRNA and protein levels in control-transfected HMECs (Fig. 4B, 4C). However, this induction was completely prevented in GILZ-transfected HMECs (Fig. 4B, 4C). TNF-induced IL-6 production was also significantly reduced by exogenous GILZ (Fig. 4E), whereas GILZ had no effect on TNF-induced IL-6 mRNA expression (Fig. 4D). These data demonstrate that HMECs respond to GILZ in a manner consistent with the response in primary HUVECs.

FIGURE 4.
FIGURE 4.

Exogenous GILZ inhibits NF-κB signaling in endothelial cells. Effect of GILZ overexpression on NF-κB activity in HMECs. NF-κB reporter construct (pNFκB-Luc) was stably coexpressed in HMECs with either pcDNA (control) or pcDNA-GILZ. (A) GILZ expression as measured by Western blot. β-actin is shown as loading control. (B–E) Expression of CCL2 and IL-6 in pcDNA (control)-transfected and pcDNA-GILZ–transfected HMECs. Data are shown for mRNA expression (B, D) and protein production (C, E) of CCL2 and IL-6 in untreated cells (NC) and following treatment with TNF (0.3, 3, and 10 ng/ml, n = 4). (F) NF-κB transcriptional activity in control or GILZ-expressing NF-κB-reporter HMECs, determined by measuring luciferase activity in the absence of treatment (NC) or following TNF treatment (0.03, 0.3, and 3 ng/ml, 4 h, n = 5). (G) NF-κB luciferase reporter activity in HMECs following infection with GILZ-expressing rAAV (or mock infection). NF-κB reporter activity was measured in untreated cells (NC) and following TNF stimulation (0.03 ng/ml, 4 h, n = 8/group). (H) Effect of NF-κB inhibition on NF-κB activity in GILZ-expressing cells. Data show NF-κB activity in pcDNA- or pcDNA-GILZ–transfected NF-κB reporter HMECs in the absence of treatment (NC), following stimulation with TNF alone (0.03 ng/ml, 4 h, n = 4), or following pretreatment with the NF-κB inhibitor, Bay 11-7082 (2 μM). Data represent mean ± SEM. *p < 0.05, **p < 0.01. ND, not detectable.

We next investigated the effects of exogenous GILZ on NF-κB activity in HMECs. TNF significantly and dose dependently increased NF-κB activity in control-transfected HMECs (Fig. 4F). However, this induction was significantly reduced in GILZ-overexpressing HMECs at all concentrations of TNF examined (Fig. 4F). To exclude the possibility that the difference in NF-κB reporter activity between the pcDNA- and pcDNA-GILZ–transfected lines stemmed from relative differences in expression of the reporter construct, we also examined the effect of GILZ expression induced by GILZ-rAAV. HMECs in which GILZ overexpression was induced via GILZ-rAAV displayed significantly reduced TNF-induced NF-κB activity relative to mock-infected cells (Fig. 4G), a finding consistent with results of stable GILZ transfection (Fig. 4F). The inhibitory effect of exogenous GILZ on NF-κB was comparable to that achieved in control HMECs using the specific pharmacological NF-κB inhibitor, Bay 11-7082 (Fig. 4H). Moreover, Bay 11-7082 treatment did not result in additional suppression of NF-κB activation in GILZ-transfected HMECs (Fig. 4H). Together, these findings provide evidence that exogenous GILZ inhibits TNF-induced NF-κB activation in endothelial cells.

Effect of exogenous GILZ on endothelial cell NF-κB p65 nuclear translocation and DNA binding

In studies in T lymphocytes, it was reported that GILZ, induced via transfection, directly binds to the NF-κB p65 subunit, and thereby inhibits its nuclear translocation and subsequent DNA binding (8, 11). However, it is unknown whether this mechanism underlies the effects of exogenous GILZ on NF-κB activation in endothelial cells. To address this, we first examined the effect of GILZ overexpression on TNF-induced p65 nuclear translocation in HMECs. Using immunohistochemistry to detect the subcellular location of p65, little p65 was detectable in nuclei of unstimulated pcDNA-transfected cells, whereas TNF induced a marked increase in nuclear p65 (Fig. 5A). In GILZ-transfected HMECs, TNF-induced p65 nuclear translocation was comparable to that in control-transfected cells (Fig. 5A), indicating that exogenous GILZ did not affect TNF-induced p65 translocation in HMECs. These findings are supported by Western blot analyses of nuclear extracts of TNF-stimulated pcDNA- and pcDNA-GILZ–transfected HMECs, which did not show any effect of GILZ overexpression on TNF-induced p65 translocation (Fig. 5B). Simultaneous assessment of the subcellular location of GILZ (Fig. 5A) revealed that, in unstimulated pcDNA-GILZ–transfected HMECs, GILZ was expressed in both the cytoplasm and the nucleus, and this was not altered by TNF treatment. These data do not support the contention that the p65 nuclear translocation is inhibited by a physical interaction with cytoplasmic GILZ. This was confirmed by immunoprecipitation experiments that failed to demonstrate a protein–protein interaction between GILZ and NF-κB p65 (data not shown).

FIGURE 5.
FIGURE 5.

Exogenous GILZ inhibits TNF-induced NF-κB (p65) DNA binding but does not affect p65 nuclear translocation in endothelial cells. (A) Expression and localization of NF-κB p65 and GILZ in pcDNA-transfected and pcDNA-GILZ–transfected HMECs, as assessed using immunohistochemistry. Cells were either not treated (NC) or treated with TNF (0.3 ng/ml, 30 min), and immunofluorescence was used to assess expression and subcellular localization of p65 (green) and GILZ (red) using confocal microscopy. DAPI-stained nuclei (blue) are also shown. Images show representative data from one of four individual experiments. Scale bar, 40μm. (B) Effect of GILZ expression on p65 nuclear translocation, as assessed by Western blot. pcDNA-transfected and pcDNA-GILZ–transfected HMECs were treated with TNF (0.03 ng/ml) for 0, 10, 20, or 30 min, and nuclear extracts were isolated and Western blotted for p65 and GILZ. TBP is also shown as loading control. Data are representative of four individual experiments. (C) Effect of GILZ expression on p65 degradation. pcDNA-transfected and pcDNA-GILZ–transfected HMECs were either not treated (NC) or stimulated with TNF (0.03 ng/ml) for 30 min. Cells were then transferred to TNF-free medium, and cells were collected after 10–40 min. Nuclear extracts were Western blotted for p65, as well as GILZ and TBP, as in (B). Data are representative of four individual experiments. (D) Effect of GILZ expression on p65 DNA binding. p65 binding to the CCL2 promoter was assessed via ChIP, in the presence or absence of TNF (0.3 ng/ml, 1 h) in pcDNA-transfected and pcDNA-GILZ–transfected HMECs (n = 3). *p < 0.05.

An alternative mechanism by which nuclear NF-κB might be regulated by exogenous GILZ is via effects on its degradation. Nuclear NF-κB is subject to constitutive proteasomal degradation via ubiquitin ligase complex (23). To address whether GILZ modulated NF-κB function in endothelial cells via this pathway, we assessed the rate of loss of immunoreactive p65 from the nucleus of TNF-activated HMECs. HMECs were treated with TNF for 30 min, after which TNF was removed, and nuclear p65 was monitored over the subsequent 40 min. In control-transfected cells, TNF-induced nuclear p65 declined to near basal levels over 40 min (Fig. 5C). In GILZ-transfected HMECs, the level of nuclear p65 was indistinguishable from that in control-transfected cells over the same time course (Fig. 5C), indicating that exogenous GILZ has no effect on nuclear p65 degradation in HMECs.

A potential mechanism for the suppressive effect of exogenous GILZ on NF-κB is inhibition of the binding of the NF-κB complex to κB sites on gene promoters. Therefore, we next examined the effect of GILZ overexpression on TNF-induced p65 DNA binding, using ChIP analysis (for primer sequences, see Table I). TNF markedly increased p65 binding to the CCL2 promoter in control cells (Fig. 5D). However, TNF induction of p65 DNA binding was almost completely abolished in GILZ-transfected cells. Similar responses were seen for the promoters of CXCL8 and VCAM-1 (data not shown). Together, these data suggest that exogenous GILZ inhibits TNF-induced NF-κB activation by suppressing the binding of p65 to target DNA sequences.

Effect of exogenous GILZ on MAPKs and MKP-1

Endothelial cell activation depends upon MAPK activation, as well as NF-κB (24), and GILZ was shown to inhibit the ERK MAPK pathway in mouse thymocytes (14). Therefore, we assessed the ability of exogenous GILZ to modulate TNF-induced MAPK phosphorylation in endothelial cells. In control-transfected HMECs, TNF induced the phosphorylation of p38, ERK, and JNK (Fig. 6A–C, 6E–G). In contrast, in GILZ-transfected cells, TNF-induced MAPK phosphorylation was markedly and significantly inhibited (Fig. 6A–C, 6E–G). This effect was achieved in the absence of an effect on the level of total MAPK expression (Fig. 6A–C). The observation that exogenous GILZ perturbed the phosphorylation of multiple MAPKs raised the possibility of an effect of GILZ on the expression of a polyspecific MAPK phosphatase. MKP-1 is known to dephosphorylate and, hence, inactivate all three MAPKs. Moreover, this molecule has been shown to have anti-inflammatory effects in endothelial cells (25–27). Therefore, we investigated the effect of GILZ overexpression on MKP-1 expression in HMECs. Compared with control-transfected cells, MKP-1 protein expression was significantly higher in GILZ-transfected cells (Fig. 6D, 6H). Taken together, our findings indicate that exogenous GILZ exerts inhibitory effects on endothelial cell adhesive function through modulation of NF-κB p65 DNA binding and MAPK activity.

FIGURE 6.
FIGURE 6.

Exogenous GILZ inhibits TNF-induced phosphorylation of MAPKs and increases MKP-1 expression. HMECs transfected with either pcDNA or pcDNA-GILZ were treated with TNF (0.03 ng/ml) for 0–30 min, and MAPK phosphorylation and MKP-1 and GILZ expression were assessed in cytoplasmic extracts via Western blot. (A–C) Representative Western blots showing phosphorylated and total p38 (A), ERK (B), and JNK (C). Densitometric data for the representative gels are shown below the blot. (D) Expression of MKP-1 and GILZ in the same samples. (E–H) Quantitative analysis of p38, ERK, and JNK phosphorylation and MKP-1 expression, as determined by densitometric analysis, expressed as a ratio of phosphorylated protein to total protein. Data were normalized to values from untreated pcDNA-transfected cells. Data represent mean ± SEM of n = 4/group. #p < 0.05, ##p < 0.01, versus corresponding untreated controls; *p < 0.05, **p < 0.01, versus corresponding pcDNA controls.

Effects of GILZ siRNA on HUVEC function

Finally, we wanted to assess the role of endogenous GILZ in controlling the responses of activated endothelial cells. Therefore, we examined the responses of HUVECs following siRNA-mediated knockdown of GILZ. As recent studies provided evidence of a role for GILZ in mediating the actions of glucocorticoids (28), we investigated the impact of GILZ deficiency on the effects of the glucocorticoid, DEX, on endothelial cell function. Initial experiments revealed that GILZ siRNA transfection reduced GILZ mRNA by almost 80% in DEX-treated cells (Fig. 7A), indicating that effective GILZ knockdown was achieved. To assess the functional contribution of GILZ, we then examined the effect of GILZ siRNA on TNF-treated HUVECs, in the presence and absence of DEX, using leukocyte rolling, transmigration, and IL-6 release as readouts. In control cells (i.e., HUVECs treated with scrambled siRNA), TNF induced increases in each of these parameters (data not shown), and DEX treatment significantly inhibited these TNF-induced increases (Fig. 7B–D). In cells transfected with GILZ siRNA, DEX retained the ability to inhibit these responses, indicating that, under experimental conditions in which endogenous GILZ is highly expressed, GILZ silencing did not affect the functional response of the endothelial cells. Similarly, GILZ siRNA did not modify the responses of cells exposed to TNF alone.

FIGURE 7.
FIGURE 7.

Effects of GILZ siRNA on TNF-induced responses in HUVECs. (A) GILZ mRNA in HUVECs transfected with either GILZ-specific or scrambled (Sc) siRNA. Data are shown for untreated cells and cells exposed to DEX (1 nM, 2 h). Data represent mean ± SEM of n = 3. *p < 0.05, **p < 0.01, versus Sc. (B–D) Responses of GILZ siRNA-transfected HUVECs in the presence of TNF, with or without DEX (used to induce GILZ). Data are shown for leukocyte rolling in response to low-dose TNF (0.03 ng/ml, 4 h) (B), leukocyte transmigration efficiency in response to high-dose TNF (1 ng/ml, 4 h) (C), and IL-6 release induced by low-dose TNF (0.03 ng/ml, 4 h) (D). The leukocyte rolling and transmigration efficiency experiments were performed in a flow chamber assay. Data represent mean ± SEM of n = 3. *p < 0.05, **p < 0.01, versus cells not treated with DEX.

Discussion

GILZ was recently reported to inhibit inflammatory responses in several animal models of inflammatory disease (11, 15). In these studies, the immunosuppressive/anti-inflammatory effects of GILZ were ascribed primarily to its effects in leukocytes, consistent with numerous in vitro observations (7, 8, 29, 30). However, our recent observation of robust GILZ expression in endothelial cells in human rheumatoid arthritis synovium raises the possibility that GILZ also has the capacity to mediate anti-inflammatory responses in this cell type (15). In this study, we demonstrate that exogenous GILZ overexpression in endothelial cells suppresses their ability to support leukocyte interactions following activation. This occurred via an inhibitory effect on NF-κB activation associated with reduced expression of adhesion molecules and chemokines. Investigation of the mechanism underlying this response revealed that, in TNF-activated endothelial cells, exogenous GILZ reduced NF-κB p65 DNA binding and inhibited MAPK activation in parallel with the induction of MKP-1. However, GILZ did not bind to NF-κB p65 or inhibit its translocation to the nucleus, as it has been reported to do in other cell types (8, 9, 31). Together, these findings indicate that exogenous GILZ has previously unrecognized anti-inflammatory actions in human endothelial cells and suggest that the mode of action of exogenous GILZ in these cells is distinct from that described in other cell types. These findings may have relevance to potential GILZ-based therapeutic approaches to inflammatory disease.

Via their ability to upregulate adhesion molecules that capture leukocytes from the mainstream of blood flow and control their entry into inflamed tissues, endothelial cells act as critical “gate-keepers” in inflammation (1). As such, modulation of this adhesive function has the potential to be an important control point in inflammatory responses. In the current study, we show that GILZ overexpression in primary HUVECs inhibits TNF-induced leukocyte rolling and adhesion under flow conditions, an effect associated with decreased expression of endothelial adhesion molecules and chemokines. The final step in leukocyte recruitment, transmigration, was also reduced by exogenous GILZ. Notably, the observed reduction in transmigration efficiency (i.e., the proportion of adherent leukocytes that subsequently transmigrate) demonstrated that this effect was not simply a reflection of the reduced leukocyte adhesion on GILZ-transfected HUVECs, but was an independent effect on transmigration. Together, these results indicate that exogenous GILZ has effects on human endothelial cells at multiple points in the sequence of interactions that mediate leukocyte recruitment from the bloodstream. Despite the breadth of these effects, the inhibitory effect of GILZ overexpression on HUVEC function, although significant, was incomplete. One potential explanation for this observation is that endothelial activation involves pathways that are not susceptible to GILZ-mediated inhibition. Alternatively, this finding may stem from the relative resistance of primary HUVECs to transfection, an interpretation supported by the much greater magnitude of effect on CCL2 and IL-6 expression observed in HMECs stably transfected with GILZ. This issue could be resolved by in vivo studies of GILZ-transgenic mice that overexpress GILZ in endothelial cells, but such mice are yet to be reported.

NF-κB is critical to the proadhesive function of activated endothelial cells, driving the expression of key genes associated with the endothelial response to inflammatory stimulation, including E-selectin, VCAM-1, CXCL8, and CCL2 (32–35). In this study, we assessed whether the NF-κB pathway is inhibited by exogenous GILZ in endothelial cells. We found that exogenous GILZ overexpression powerfully inhibited NF-κB p65 transcriptional activity in HMECs, an effect associated with reduced expression of CCL2 and IL-6. The effects of exogenous GILZ were comparable to those of BAY 11-7082, a potent and specific NF-κB inhibitor; moreover, this inhibitor failed to further reduce NF-κB function in GILZ-transfected cells. These findings indicate that, as was reported for various leukocyte populations, NF-κB is a key target by which exogenous GILZ mediates anti-inflammatory effects in endothelial cells. In this study, we were unable to replicate NF-κB reporter experiments in HUVECs, because in these primary cells, double transfection was not compatible with the technique used to induce GILZ. However, the observation that, in HUVECs, exogenous GILZ significantly reduced the expression of key NF-κB target genes at the transcriptional level (Figs. 2, 3) provides support for the concept that this mechanism is also active in primary HUVECs.

Studies of murine thymocytes found that GILZ inhibits NF-κB function by preventing its nuclear translocation and subsequent DNA binding (8). Evidence from GILZ/p65-cotransfected cells suggests that this stems from a direct interaction between GILZ and p65 in the cytoplasm. In addition, it was reported more recently that GILZ homodimerization via the proline-rich region of the C-terminal in the GILZ protein was necessary for this GILZ/NF-κB interaction (9). In contrast to previous studies, we observed that p65 nuclear translocation was not inhibited in GILZ-transfected endothelial cells, and GILZ did not interact directly with p65 or alter its subcellular localization in response to activation. These findings suggested that suppression of NF-κB transcriptional activity in endothelial cells mediated by exogenous GILZ does not depend on a protein–protein interaction that prevents p65 nuclear translocation. However, ChIP experiments indicate that exogenous GILZ inhibited p65 binding to the κB site on the CCL2, CXCL8, and VCAM-1 promoters. The exact mechanism through which GILZ impacts on DNA binding remains unclear. However, it is known that posttranslational modifications (e.g., phosphorylation and acetylation) of NF-κB are absolutely required for its optimal transcriptional activity (36–38), and, in many cases, these posttranslational modifications are able to alter transcriptional activity without affecting nuclear translocation (39–41).

The present data also indicate that exogenous GILZ inhibits activation of endothelial cell ERK, p38, and JNK MAPKs. GILZ was found to inhibit TNF-induced ERK activation in T cells (13). However, the effects of GILZ on p38 and JNK activation have not been reported. The present results indicate that, in endothelial cells, transfected GILZ has a broad inhibitory effect on the activation of MAPKs. Interestingly, it has been observed that MAPK activation facilitates NF-κB activation in human endothelial cells (42). As such, these findings raise the possibility that inhibition of MAPK activation represents a pathway by which exogenous GILZ suppresses NF-κB function in activated endothelial cells. In parallel, we observed that GILZ overexpression increases MKP-1 expression in these cells. MKP-1 belongs to the dual-specificity phosphatase family, and it functions to dephosphorylate and inactivate MAPKs (25). Although best recognized as an inhibitor of p38 and JNK MAPKs, MKP-1 has also been observed to inhibit ERK MAPKs in various contexts (43, 44). In endothelial cells, MKP-1 was shown to suppress activation and adhesion molecule expression induced by a range of proinflammatory stimuli, including TNF (26, 27, 45). Moreover, recent studies demonstrate that induction of MKP-1 expression can lead to inhibition of NF-κB activation (46). Therefore, the elevated expression of MKP-1 in GILZ-transfected HMECs observed in this study suggests this effect as a novel intermediary contributing to the GILZ-dependent inhibition of NF-κB activation in endothelial cells.

To complement the studies of GILZ-transfected cells, we also examined the effect of removing GILZ from activated endothelial cells, testing the hypothesis that the absence of endogenous GILZ would exacerbate endothelial responses to inflammatory stimuli. To facilitate detection of a functional role for endogenous GILZ, we examined cells treated with DEX, an agent that strongly induces GILZ expression. Under these conditions, removal of GILZ did not change the functional response of HUVECs to TNF-mediated stimulation. Similarly, the absence of GILZ did not alter the capacity of the glucocorticoid DEX to inhibit endothelial responses to TNF. These findings indicate that endogenous GILZ, at least under these conditions, does not inhibit endothelial cell activation. These findings are paralleled by findings from our recent examination of GILZ-deficient mice, in which the absence of endogenous GILZ did not alter the inflammatory response in a range of inflammatory models (47). Notably, however, in this in vivo study, exogenously delivered GILZ was able to inhibit inflammation in collagen-induced arthritis, mirroring the inhibitory effects of GILZ on endothelial cell activation observed in the current study. Similar observations were made in primary human synovial fibroblasts, in which exogenous GILZ was able to inhibit cytokine expression (15) but GILZ silencing was without effect (47). At this point, the reason for the contrast between the actions of endogenous and exogenous GILZ is unclear. Nevertheless, from a therapeutic viewpoint, the present findings demonstrate that, in addition to its effects in vivo, exogenously administered GILZ modulates inflammatory activation of endothelial cells in vitro.

In conclusion, to our knowledge, this study provides the first demonstration that exogenous GILZ mediates anti-inflammatory functions in human endothelial cells. GILZ overexpression strongly inhibits NF-κB p65 DNA binding and transcriptional activity, but these effects on endothelial cells appear to be independent of binding to p65 or inhibition of p65 nuclear translocation. In addition, we show that exogenous GILZ inhibits ERK MAPK, as well as p38 and JNK, in these cells, effects associated with induction of the expression of MKP-1. Together, these results demonstrate previously unrecognized effects of GILZ in human endothelial cells. Moreover, given the pivotal role of endothelial cell activation in inflammation, these findings raise the possibility that induction of endothelial GILZ expression could represent a novel therapeutic modality in human inflammatory disease.

Disclosures

The authors have no financial conflicts of interest.

Acknowledgments

We thank Joanne Mockler and Prof. Euan Wallace (Department of Obstetrics and Gynaecology, Monash University) for assistance with umbilical cord collections, Dr. Carlo Riccardi for provision of the human GILZ construct, and Drs. Scott Loiler and Margriet Vervoordeldonk for provision of GILZ-rAAV.

Footnotes

  • This work was supported by funding from the National Health and Medical Research Council of Australia, Arthritis Australia, and the Heart Foundation of Australia. M.J.H. is a National Health and Medical Research Council Senior Research Fellow.

  • Abbreviations used in this article:

    ChIP
    chromatin immunoprecipitation
    DEX
    dexamethasone
    GILZ
    glucocorticoid-induced leucine zipper
    HMEC
    human microvascular endothelial cell
    rAAV
    recombinant adeno-associated virus 5
    siRNA
    small interfering RNA
    TBP
    TATA-binding protein.

  • Received September 24, 2012.
  • Accepted April 30, 2013.

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